The polysaccharide capsules of Streptococcus pneumoniae represent the single most important virulence factor of this organism. Structurally and antigenically diverse, 90 serotypes have been recognized. Biosynthesis of these polysaccharides involves at least two distinct mechanisms. One, observed for the type 3 polysaccharide (-3)-beta-D-GlcUA-(1,4)-beta-D-Glc-(1-), involves a single processive enzyme (the type 3 synthase) that is responsible for generating both glycosidic linkages and for transporting the polymer out of the cell. The type 3 synthase is a member of a family of glycosyltransferases that includes the streptococcal and eukaryotic hyaluronan synthases, chitin synthases, and cellulose synthases. Our work with the type 3 synthase has shown that synthesis initiates on a lipid primer. Synthesis occurs by a processive mechanism under high substrate concentrations. Under low substrate concentrations, however, the synthase functions as a nonprocessive enzyme. When only a single substrate is present, chain termination and release of the polysaccharide result. Synthase stability in S. pneumoniae appears to be dependent on substrate levels, thus suggesting another means of controlling synthase function. The mechanism of synthesis of most other S. pneumoniae capsular polysaccharides is similar to that of some LPS O-antigens, involving initiation and transport of subunits across the membrane on a lipid carrier, followed by polymerization. Homologous proteins (CpsABCD) involved in this type of biosynthesis occur in S. pneumoniae, Rhizobium, other streptococci, and staphylococci. We have demonstrated an interaction among the S. pneumoniae proteins, and we hypothesize that they function to enhance polymerase activity. The goals of the proposed research are to characterize the S. pneumoniae polymerases and the factors that directly influence their functions. We will address these goals by: 1) identifying the primer for type 3 synthesis and reconstituting the system; 2) identifying functional domains/residues of the type 3 synthase; 3) characterizing synthase functions and polysaccharide release in vivo; and 4) characterizing functions common to the synthesis of non-type 3 capsular polysaccharides. The results of these studies will provide insights into mechanisms of polysaccharide synthesis that are shared among many prokaryotic and eukaryotic systems.